The FLRT3-UNC5B checkpoint pathway inhibits T cell–based cancer immunotherapies

Cancers exploit coinhibitory receptors on T cells to escape tumor immunity, and targeting such mechanisms has shown remarkable clinical benefit, but in a limited subset of patients. We hypothesized that cancer cells mimic noncanonical mechanisms of early development such as axon guidance pathways to evade T cell immunity. Using gain-of-function genetic screens, we profiled axon guidance proteins on human T cells and their cognate ligands and identified fibronectin leucine-rich transmembrane protein 3 (FLRT3) as a ligand that inhibits T cell activity. We demonstrated that FLRT3 inhibits T cells through UNC5B, an axon guidance receptor that is up-regulated on activated human T cells. FLRT3 expressed in human cancers favored tumor growth and inhibited CAR-T and BiTE + T cell killing and infiltration in humanized cancer models. An FLRT3 monoclonal antibody that blocked FLRT3-UNC5B interactions reversed these effects in an immune-dependent manner. This study supports the concept that axon guidance proteins mimic T cell checkpoints and can be targeted for cancer immunotherapy.


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Figs. S1 to S17 Table S1 Fig. S1: Phylogenetic map of axon guidance-like genes.Genes with axon guidance related activities were identified by literature and keyword searches.Identified genes were assessed based on phylogeny and displayed in a circular phylogenetic map.

Fig. S2:
Axon guidance molecule expression in cancer.Genes with axon guidance related activities were identified by literature and keyword searches.The expression of 177 axon guidance genes from 8431 cancer patients belonging to 34 different cancers were queried using TCGA.Mean expression for each gene were calculated in each of the cancers.A heatmap with two-way hierarchical clustering was generated using gplots v3.1.0library in R v4.1.0using heatmap.2function.
Color key Value Supplemental Figure 3 Fig.S3: Effect of FLRT3 transfection on proliferation and viability of 293T cells in vitro.(A and B) 293T-OKT3 cells were transiently transfected with EV, FLRT3 and B7-1 genes and 6 days later (A) cell number and (B) % viability were determined by trypan blue method.(C) CFSE labelled human PBMCs from 3 healthy donors were co-cultured with 293T-OKT3 cells transfected with FLRT3, PD-L1 and B7-1 genes followed by analysis of proliferation by CFSE dilution.Quantification of proliferating CD4+ T cells is shown for one representative donor.(D) Summary data quantifying % change in proliferation in FLRT3 group compared to EV control for CD8+ and CD4+ T cells separately for 3 donors.For (A to C) statistical significance was determined by One-way ANOVA with Tukey's post-hoc test for multiple comparisons.Supplemental Table 1 Table S1.FIND-IO screening results from 4 independent screens of normal, healthy donors.CD8+ and CD4+ T cell proliferation based on percent CFSE divided cells for axon guidance-related proteins transfected and expressed as proteins on 293T-OKT3 cells.293T-OKT3 cells were UV-irradiated and co-cultured with primary total PBMCs (cryo-recovered for fresh) or primary enriched T cells for 72 hours, followed by assessment of T cell proliferation by flow cytometry.Proliferation values were converted to a rank from 1 to 57 for each screen and then averaged for a combined rank score for CD8+ and CD4+ T cells Fig. S4: UNC5B is expressed on activated human T cells.(A and B) Human PBMCs from 3 healthy donors were analyzed for expression of FLRT3's binding partners LPHN1, LPHN3, UNC5A-D on B and T cells (A) monocytes (B) on day 0. (C to F) Human PBMCs from 3 healthy donors were activated by co-culturing with 293T-OKT3 (C and D) or with soluble OKT3 (E and F) and expression of FLRT3's binding partners LPHN1, LPHN3, UNC5A-D was measured on T cells on day 3 and 4 (activated).CD8+ (C and E) and CD4+ T cells (D and F) data are shown separately.(G) Human PBMCs from 2 healthy donors were activated with soluble OKT3 for 10 days and UNC5B expression was analyzed along with other checkpoint receptors PD-1, TIM-3, LAG-3.Quantification of PD-1+, TIM-3+, LAG-3+ cells within the UNC5B+ population is shown.Each data point represents one donor and error bars denote + Range in bar graphs.

Fig. S16 :
Fig. S16: Working model of FLRT3-UNC5B mediated immune co-inhibitory mimicry.FLRT3 expression on tumor cells in cancer functions as a mimic of traditional coinhibitory ligands when it binds to UNC5B that is aberrantly upregulated on Ag-stimulated T cells.UNC5B signaling is capable of inhibiting TCR-CD3 signaling that prevents T cell activation and recruitment and/or infiltration to the tumor microenvironments.

Fig. S17 :
Fig. S17: Gating strategies.Gating strategies used to evaluate CD8+ and CD4+ T cell for proliferation in AGM screening (A), assessment of FLRT3 binding partner expression (B), assessment of UNC5B, PD-1, TIM-3 and LAG-3 (C) and ex vivo analysis from the spleens of Ctrl Fc or FLRT3-Fc treated mice (D).